Microorganism

ABSTRACT

A microorganism which is Rhodococcus rhodochrous strain NCIMB 41164 or a mutant thereof. A method of culturing the microorganism in a culture medium comprising urea or urea derivative is claimed. A nitrite hydratase obtainable from the microorganism is claimed. Also claimed is a process of preparing an amide from the corresponding nitrite wherein the nitrite is subjected to a hydration reaction in an aqueous medium in the presence of a biocatalyst selected from the group consisting of a microorganism which is a Rhodococcus rhodochrous strain NUMB 41164, a mutant thereof and a nitrite hydratase obtainable from Rhodococcus rhodochrous strain NCIMB 41164 or a mutant thereof. Also claimed is a method of storing the Rhodococcus rhodochrous NUMB 41164.

The present invention relates to a microorganism and to methods ofculturing and storing the microorganism. The invention also relates to anovel nitrile hydratase enzyme and also to a method of converting anitrile to an amide employing the nitrile hydratase enzyme.

It is well known to employ biocatalysts, such as microorganisms thatcontain enzymes, for conducting chemical reactions. Nitrile hydrataseenzymes are known to catalyse the hydration of nitriles directly to thecorresponding amides. Typically nitrile hydratase enzymes can beproduced by a variety of microorganisms, for instance microorganisms ofthe genus Bacillus, Bacteridium, Micrococcus, Brevibacterium,Corynebacterium, Pseudomonas, Acinetobacter, Xanthobacter, Streptomyces,Rhizobium, Klebsiella, Enterobacter, Erwinia, Aeromonas, Citrobacter,Achromobacter, Agrobacterium, Pseudonocardia and Rhodococcus.

Many references have described the synthesis of nitrile hydratase withinmicroorganisms. Amaud et al., Agric. Biol. Chem. 41: (11) 2183-2191(1977) describes the characteristics of an enzyme they refer to as‘acetonitrilase’ in Brevibacterium sp R312 which degrades acetonitrileto acetate via the amide intermediate. Asano et al., Agric. Biol. Chem.46: (5) 1183-1189 (1982) isolated Pseudomonas chlororaphis B23 whichproduced nitrile hydratase to catalyse the conversion of acrylonitrileto acrylamide, generating 400 g/L acrylamide. The article by Yamada etal., Agric. Biol. Chem. 50: (11) 2859-2865 (1986) entitled, “Optimumculture conditions for production by Pseudomonas chlororaphis B23 ofnitrile hydratase”, considered the optimisation of the medium componentsof the growth medium, including the inducer added for nitrile hydratasesynthesis. Methacrylamide was found to be the best inducer for thisorganism. Methacrylamide was included in the culture at the start ofgrowth. Various strains of the Rhodococcus rhodochrous species have beenfound to very effectively produce nitrile hydratase enzyme.

EP-0 307 926 describes the culturing of Rhodococcus rhodochrous,specifically strain J1 in a culture medium that contains cobalt ions. Aprocess is described for biologically producing an amide in which anitrile is hydrated by the action of a nitrile hydratase produced byRhodococcus rhodochrous J1, which has been cultured in the presence ofcobalt ion. The use of various inducers (including crotonamide) isdescribed for the synthesis of nitrile hydratase. In one embodiment anamide is produced in a culture medium of the microorganism in which asubstrate nitrile is present. In another embodiment a substrate nitrileis added to the culture medium in which a nitrile hydratase has beenaccumulated to conduct the hydration reaction. There is also adescription of isolating the microbial cells and supporting them in asuitable carrier, for instance by immobilisation, and then contactingthem with a substrate. The nitrile hydratase can be used to hydratenitriles into amides, and in particular the conversion of3-cyanopyridine to nicotinamide.

EP-0 362 829 describes a method for cultivating bacteria of the speciesRhodococcus rhodochrous comprising at least one of urea and cobalt ionfor preparing the cells of Rhodococcus rhodochrous having nitrilehydratase activity. Specifically described is the induction of nitrilehydratase in Rhodococcus rhodochrous J1 using urea or urea derivativeswhich markedly increases the nitrile hydratase activity. Urea or itsderivatives are added to the culture medium in one batch at a time orsequentially and cultivation occurs over 30 hours or longer, forinstance up to 120 hours. An article by Nagasawa et al., Appl.Microbiol. Biotechnol. 34: 783-788 (1991), entitled “Optimum cultureconditions for the production of cobalt-containing nitrile hydratase byRhodococcus rhodochrous J1”, describes isolating J1 as an acetonitrileutilising strain which synthesises two different nitrile hydratases anda nitrilase depending upon the culture conditions used. One nitrilehydratase is induced optimally by urea and urea analogues. Urea is addedat the start of the culturing process and seems to become efficient asan inducer only when the basal medium is nutrient rich. Induction of theenzyme started gradually and increased in growth until it reached amaximum after 5 days of cultivation. The activity was found to decreaseon prolonged cultivation.

Rhodococcus rhodochrous J1, is also used commercially to manufactureacrylamide monomer from acrylonitrile and this process has beendescribed by Nagasawa and Yamada Pure Appl. Chem. 67: 1241-1256 (1995).

Leonova et al., Appl. Biochem. Biotechnol. 88: 231-241 (2000) entitled,“Nitrile Hydratase of Rhodococcus”, describes the growth and synthesisof nitrile hydratase in Rhodococcus rhodochrous M8. The nitrilehydratase synthesis of this strain is induced by urea in the medium, theurea also acting as a nitrogen source for growth by this organism.Cobalt is also required for high nitrile hydratase activity. Thisliterature paper looks at induction and metabolic effects in the main.

Leonova et al., Appl. Biochem. Biotechnol. 88: 231-241 (2000) alsostates that acrylamide is produced commercially in Russia usingRhodococcus rhodochrous M8. Russian patent 1731814 describes Rhodococcusrhodochrous strain M8.

Rhodococcus rhodochrous strain M33 that produces nitrile hydratasewithout the need of an inducer such as urea is described in U.S. Pat.No. 5,827,699. This strain of microorganism is a derivative ofRhodococcus rhodochrous M8.

The production of acrylamide monomer in particular is desirable via thebiocatalytic route. In the review publication by Yamada and Kobayashi,Biosci. Biotech. Biochem. 60: (9) 1391-1400 (1996) titled “NitrileHydratase and its Application to Industrial Production of Acrylamide” adetailed account of the development of a biocatalytic route toacrylamide is described. Three successively better catalysts and theircharacteristics for acrylamide production and in particular the thirdgeneration catalyst Rhodococcus rhodochrous J1 are described in somedetail.

A major disadvantage with the use of biocatalysts is the general lack ofstability observed with wet microbial material during storage,transportation and use. Even with relatively stable enzymes and bacteriasuch as nitrile hydratases in Rhodococcal cells, the potential forspoilage before use has led to acceptance within the industry for theneed to process the biocatalyst cell suspension in some way e.g. byfreezing or freeze-drying of the aqueous mixture or alternativelyimmobilisation of the cells in some polymer matrix. In order to achievemaximum productivity from the biocatalyst it is important that themaximum biocatalytic activity is retained during its preparation andstorage prior to use. In Chaplin and Bucke (1990) In: Enzyme Technology,published by Cambridge University Press, p 47 (Enzyme preparation anduse) it was recognised that enzyme inactivation can be caused by heat,proteolysis, sub optimal pH, oxidation denaturants and irreversibleinhibitors. A number of substances may cause a reduction in the rate ofan enzymes ability to catalyse a reaction. This includes substances thatare non-specific protein denaturants, such as urea.

In the presentation, Protein Stability, by Willem JH van Berkel,Wageningen University, factors that may cause deactivation or unfoldingwere considered and these included proteases, oxidation due to thepresence of oxygen or oxygen radicals and denaturing agents causingreversible unfolding, such as urea.

Chaplin and Bucke (1990) In Enzyme Technology, published by CambridgeUniversity Press, p73 (Enzyme preparation and use) revealed that the keyfactor regarding the preservation of enzyme activity involvesmaintaining the conformation of the enzyme structure. Therefore it wasconsidering important to prevent unfolding, aggregation and changes inthe covalent structure. Three approaches for achieving this wereconsidered: (1) use of additives; (2) the controlled use of covalentmodification; and (3) enzyme immobilisation.

EP-B-0 243 967 describes the preservation of nitrile hydration activityof nitrilase by the addition of stabilizing compounds selected fromnitriles, amides and organic acids and their salts, to a solution orsuspension of the enzyme or the immobilized form of the enzyme. Itclearly states in the description that while a solution or suspension ofa microorganism capable of producing nitrilase that hydrates nitrilessuch as acrylonitrile, to produce the corresponding amides such asacrylamide may be stored at room temperature as long as the storageperiod is short, storage at a low temperature, especially at atemperature in the vicinity of 0° C. is preferred. It was described inEP-A-0 707 061 that addition of inorganic salts at a concentration ofbetween 100 mM to the saturation concentration of the inorganic salts toan aqueous medium containing either a suspension of microbial cells orimmobilized microbial cells, preserved the cells and enzyme activity fora prolonged period of time. This technique is described for thepreservation of microbial cells that have nitrile hydratase or nitrilaseactivity. The addition of bicarbonate or carbonate salts to an aqueoussolution of immobilized or unimmobilised microbial cells havingnitrilase activity is described in U.S. Pat. No.-B-6,368,804.Immobilisation has frequently involved removal of the enzyme from thewhole cell, before immobilising the enzyme in a matrix. However,although such immobilisation provides very good protection for theenzyme, extraction of the enzyme from the whole cell is an intricatestep, which can be time-consuming, expensive and can lead to loss ofenzyme. Additionally whole microbial cells can be immobilized. U.S. Pat.No. 5,567,608 provides a process of immobilising whole cell biocatalystin a cationic copolymer which has good storage stability and preventsputrefaction.

Rhodococcus rhodochrous J1, which is used commercially to manufactureacrylamide monomer, is immobilised to (a) allow transportation and (b)to increase the longevity of the biocatalyst in use. In U.S. Pat. No.5,567,608 the inventors state that biocatalysts are normally immobilizedfor use on an industrial scale, to facilitate ease of separation of thebiocatalyst from the reaction product, preventing impurities from thebiocatalyst eluting into the product and to assist in continuousprocesses and recycling of the biocatalyst. However, immobilisation isan extra processing step that requires an additional plant and the useof potentially a number of other raw materials such as alginate,carrageenan, acrylamide and other acrylate monomers, and vinyl alcohol.Thus, this is an expensive processing step.

Various other ways have been proposed for minimising the deleteriouseffects of enzyme inactivation in an attempt reduce the negative impacton a chemical reaction process.

It is also known to freeze dry biocatalysts in order to preserve theactivity of an enzyme in storage over a prolonged period of time. Againthis is a potentially expensive processing step that is normally carriedout with biocatalysts prepared on a small scale. Cryopreservation inliquid nitrogen or in the vapour phase of liquid nitrogen also affordslong-term storage of microbial cells but requires a constant supply ofliquid nitrogen. Freezing of recovered biomass or semi-pure or pureenzymes at temperatures of <−18° C. is also known to preservebiocatalytic activity for prolonged periods of time.

Furthermore, once the cell mass is introduced to the reactor and thereaction is taking place minimisation of the loss of efficacy iscritical to the operational efficiency and the process economics. Onceagain, immobilisation of the microbial cells into some polymer matrix isstandard procedure to optimise these process parameters.

It would therefore be desirable to provide a process and a biocatalystwhere these disadvantages can be overcome.

According to the present invention we provide a microorganism that isRhodococcus rhodochrous strain NCIMB 41164 or a mutant thereof.

This new microorganism has been found to readily produce nitrilehydratase. We have found that this new microorganism (and the nitrilehydratase produced therefrom) can be used in a process of convertingnitriles, to the amide. Rhodococcus rhodochrous NCIMB 41164 isparticularly of use for the conversion of (meth)acrylonitrile to(meth)acrylamide. The microorganism and enzyme have been found to remainactive, and in some cases even increase in activity, over long periodsof time and furthermore can be recovered from the reaction mixture withundiminished activity after preparation of acrylamide at >50% w/w. Thusit can be, if required, reused either directly or after a further periodof storage.

The details of the new strain Rhodococcus rhodochrous NCIMB 41164 aregiven below:

1. Origin and Deposition

The Rhodococcus rhodochrous strain was isolated by us from soil inBradford, England and deposited on Mar. 5, 2003 at the NationalCollection of Industrial and Marine Bacteria (NCIMB), where it wasassigned the accession number NCIMB 41164 under the Budapest Treaty.

2.Taxonomic Identification of the Microorganism

Identification of the soil isolate was carried out using the techniqueof 16S rDNA analysis. The sequence of the 16S rDNA gene obtained fromthe soil isolate was compared with nucleic acid sequence databases. Thesequence obtained was compared to those found in a proprietary database(Microseq™) and the top 20 hits were determined. Comparison of thesequence with this database identified the best match as Rhodococcusrhodochrous with a 97.48% similarity. This is a genus level match, butwas most likely to be a strain of Rhodococcus rhodochrous. A furthersearch search against the public EMBL database identified the best matchfor this database to Rhodococcus rhodochrous with 99.698% similarity.

3. Morphological and Cultural Characteristics

-   -   (1) Polymorphic growth    -   (2) Motility: immotile    -   (3) Non-spore former    -   (4) Gram positive    -   (5) Aerobic    -   (6) Growth on nutrient agar gives salmon pink round colonies        within 48 hours at 30° C.        4. Cultivation and Nitrile Hydratase Synthesis

The Rhodococcus rhodochrous NCIMB 41164 of the present invention can becultured under any conditions suitable for the purpose in accordancewith any of the known methods, for instance as described in theaforementioned prior art. Preferably the microorganism is cultured in aculture medium that comprises urea or a derivative of urea. We havefound that this microorganism can be grown in a medium containingacetonitrile or acrylonitrile as an inducer of the nitrile hydratase. Inthe presence of urea or urea derivative as an inducer and cobaltchloride as a source of cobalt ions, very high nitrile hydrataseactivity is achieved. For example urea and cobalt are added to themedium described in the experimental examples.

Desirably the Rhodococcus rhodochrous NCIMB 41164 can be cultured togive high enzyme activity, for instance about 250-300,000 μmol min−¹/gdry biomass at 15° C. High nitrile hydratase activity can be achieved ifurea or a urea derivative is present in the culture medium. It may bepresent at the start of the culture or it may be added at some pointduring growth, but generally should be added before the onset of thestationary phase of growth. High nitrile hydratase activity canpreferably be achieved if urea or the urea derivative is not present inany substantial amount in the culture medium at the start of themicroorganism growth but is introduced later. By this we mean that ureaor the urea derivative is not present or is present in an amount of lessthan 0.2 g/l, preferably less than 0.1 g/l. More preferably the culturemedium is substantially free (i.e less than 0.2 g/l) of urea or the ureaderivative for at least the first six hours of microorganism growth. Itis especially preferred if the growth medium of the microorganism issubstantially free of urea or the urea derivative for at least 12 hoursand in some cases at least 24 hours before the introduction of the ureaor the urea derivative as the growth rate of the microorganism is higherin the absence of urea or the urea derivative, but that it is addedbefore 48 hours culturing of the microorganism. We have found that thisenables higher nitrile hydratase activity to occur in a shorter periodof time than if the urea or the urea derivative had been added at thestart of culturing.

The invention also relates to a nitrile hydratase obtainable from amicroorganism which is Rhodococcus rhodochrous NCIMB 41164 or a mutantthereof.

A further aspect of the invention concerns a process of preparing anamide from the corresponding nitrile wherein the nitrile is subjected toa hydration reaction in an aqueous medium in the presence of abiocatalyst selected from the group consisting of a microorganism whichis Rhodococcus rhodochrous NCIMB 41164, a mutant thereof and a nitrilehydratase obtainable from Rhodococcus rhodochrous NCIMB 41164 or amutant thereof. Hereafter the term ‘biocatalyst’ refers to the nitrilehydratase that is synthesised within the Rhodococcus rhodochrous NCIMB41164 cell and may include the Rhodococcus rhodochrous NCIMB 41164 cellitself. Thus, the biocatalyst could be used as a whole cell preparationin a fermentation medium, as an aqueous suspension, as a recovered cellpaste as an immobilized cell preparation or as any other form of thenitrile hydratase suitable for the conversion of nitrile to amide thatsatisfies the requirements of this invention.

This process is particularly suitable for readily preparing an amidefrom the corresponding nitrile. In particular aqueous solutions of amidecan be prepared in high concentration. The process is especiallysuitable for preparing acrylamide or methacrylamide.

The biocatalyst may be used as a whole cell catalyst for the generationof amide from nitrile. It may be immobilised for instance entrapped in agel or it may be used preferably as a free cell suspension.Alternatively the nitrile hydratase enzyme may be extracted and forinstance used directly in the process of preparing the amide.

In one preferred way of carrying out the process the biocatalyst isintroduced into an aqueous medium suitable for carrying out theculturing of the microorganism. Typically a suspension of thebiocatalyst, for instance whole cells of the microorganism, may beformed. A nitrile, for instance acrylonitrile or methacrylonitrile isfed into the aqueous medium comprising the biocatalyst in such a waythat the concentration of (meth) acrylonitrile in the aqueous medium ismaintained at up to 6% by weight. Nitrile such as acrylonitrile ormethacrylonitrile is more preferably fed into the reaction medium andthe reaction allowed to continue until the concentration of amide, forinstance acrylamide or methacrylamide reaches the desired level, inparticular between 30 and 55% by weight. Most preferably theconcentration is around 50% by weight.

This new strain of Rhodococcus rhodochrous (NCIMB 41164) is capable ofproducing aqueous acrylamide solutions in high concentration (forinstance 50% acrylamide). Desirably the reaction may be carried out as afree cell process using a fed-batch type reactor to which thebiocatalyst (Rhodococcus rhodochrous NCIMB 41164) is added in the formof fermentation broth or as harvested biomass.

The activity of the biocatalyst (Rhodococcus rhodochrous NCIMB 41164)and the nitrile hydratase produced therefrom is such that it can berecycled and reused for further hydration of nitrile to thecorresponding amide.

Recycling of the biocatalyst is particularly suitable for any case ofconverting (meth) acrylonitrile to (meth) acrylamide. Thus in themanufacture of acrylamide when the reaction process is complete andacrylamide has been produced at the appropriate concentration, thecatalyst can be removed and re-used to produce another batch ofacrylamide without loss in nitrile hydratase activity. This can even beachieved after the biocatalyst has been stored in water for several days(for instance three days) prior to reuse. It is even possible to preparea third batch of acrylamide, even after further storage.

According to one aspect of the invention we provide an aqueouscomposition comprising a biocatalyst that is or is obtainable from themicroorganism Rhodococcus rhodochrous strain NCIMB 41164 or a mutantthereof and wherein the biocatalyst is in the form of a non-activelygrowing free cell microorganism. We also provide a method of storing thebiocatalyst, that is in the form of a non-actively growing free cellmicroorganism.

The microbial cells of the biocatalyst used to carry out the conversionof nitrile to amide, may be regarded as a non-actively growing culture.By this we mean that the medium and the storage conditions in which themicroorganism is held would not be expected to promote growth. Thestorage medium can for instance be the Rhodococcus rhodochrous NCIMB41164 cells that maybe recovered from the fermentation medium. Or thecells maybe used directly in the fermentation medium, or they maybepresent as an aqueous suspension in a suitable suspending medium forinstance; water; physiological saline solution; a suitable buffersolution such as phosphate buffer or any other similar buffer or agrowth medium where metabolism in the microorganism cells issubstantially zero as determined by measuring the growth rate, or thebiomass concentration or oxygen consumption or nutrient consumption, orother form of measurement generally used to monitor microbial growth andmetabolism.

The composition or the storage medium may comprise any residualfermentation broth components. The fermentation broth may include any ofthe typical ingredients used for culturing the microorganism and alsomay include products and by-products produced by the microorganism.Typical components of the fermentation broth include sugars,polysaccharides, proteins, peptides, amino acids, nitrogen sources,inorganic salts, vitamins, growth regulators and enzyme inducers.Specifically this could include monosaccharides or disaccharides assugars; ammonium salts or other nitrogen sources; inorganic salts suchas phosphates, sulphates, magnesium, calcium, sodium and potassiumsalts; metal compounds; vitamins; and complex fermentation mediumcomponents, for example corn steep liquor; peptone; yeast extract;organic or inorganic compounds that may be used for specific microbialgrowth requirements; specific enzyme inducers (such as urea that is usedto induce the nitrile hydratase of Rhodococcus rhodochrous NCIMB 41164);and organic acids such as citrate or pyruvate; and any other organic orinorganic compounds that may be required to ensure successful growth ofthe Rhodococcus rhodochrous NCIMB 41164.

Usually when a biocatalyst, such as one that produces nitrile hydratase,is stored without continued growth for a period of time, even for a fewdays, it is normal to remove the microbial cells from the fermentationbroth, whether it is the cells that are required as the catalyst, orwhether the enzyme is recovered from the the cells or fermentationmedium. This is to prevent microbial growth in the fermentation brothcausing putrefaction of the broth and to reduce protease activity thatcan cause the breakdown of the enzyme that is required. It is normaltherefore to preserve the fermentation broth per se or to remove thecells to prevent the degradation of the biocatalyst through extraneousbiological activity such as microbial contamination. The biocatalyticactivity could normally be expected to reduce in a very short period oftime such as within a day and certainly in less than two days if thiswere not carried out.

Methods of preserving the activity during the storage of biocatalysts,even for periods of time up to one-week, have normally involved removalof the biocatalyst from the fermentation broth and/or immobilisation ofthe biocatalyst in a suitable matrix and/or stabilisation usingstabilising substances which then either become contaminants in thereaction mixture and this may be a problem further downstream or anadditional processing step is required to remove the stabilizingcompound or additive from the microbial cell suspension before it isused as a biocatalyst.

In the absence of such preservation treatments and normally biocatalyststhat are kept at ambient temperatures tend to lose activity to theextent that they are no longer as effective or even suitable forcatalysing reactions.

Growth of a microorganism for use as a biocatalyst may take place over aperiod of several days. During this time the microorganism is activelygrowing, that is to say balanced growth where the biomass is increasingtogether with an increase in and maintenance of the overall chemicalcomposition of the cell.

Normally the growth of microorganisms is limited either by theexhaustion of nutrient or the accumulation of toxic products ofmetabolism and the growth rate reduces. Growth is maintained by feedingappropriate nutrients and maintaining a correct temperature and pH forgrowth and where required supplying oxygen.

The storage method described here promotes effective stability such thatthe biocatalyst can be readily used without any significant loss inactivity. Storage stability is achieved without the necessity ofresorting to for instance immobilisation, addition of stabilizingcompounds or freeze drying. Storage stability may be achieved withoutresorting to removal of any of the fermentation broth components such asurea or urea derivatives, even though urea is a known proteindeactivator.

The composition or the environment used in the method of storage maycontain oxygen or can be a substantially oxygen free environment. Byoxygen free we mean that the concentration of oxygen should be less than1% dissolved oxygen concentration Removal of oxygen from thefermentation broth can be achieved by any of the conventional methodsfor removing oxygen. These include purging for a period of time with aninert gas, removal of any head-space in the storage container, storingunder diminished pressure or the addition of known oxygen scavengerssuch as ascorbic acid or hydrazine and hydrazide.

It would have been expected that after 2 days and especially afterseveral days storage there would be some loss in nitrile hydrataseactivity. This would have been expected even in the absence of oxygen.It would have been expected especially in the presence of residualfermentation broth components, such as urea, and also at temperatures ofabove 0° C. This is because protease enzymes in the biocatalyst might beexpected to break down other proteins in the cell, including the nitrilehydratase. Furthermore, the presence of urea or urea derivative could beexpected to be detrimental, since urea is known to be a proteindeactivator. However, the biocatalyst suffers none of the expecteddisadvantages and thus suffers no significant loss in nitrile hydrataseactivity.

On the contrary we find that during the storage period the activity ofthe biocatalyst comprising nitrile hydratase can in some cases actuallyincrease. Thus in another aspect of the invention we provide a method ofincreasing the nitrile hydratase activity of a biocatalyst capable offorming nitrile hydratase by storing the biocatalyst in a storage mediumin accordance with the storage method of the present invention.Therefore, the method can result in a new biocatalyst composition byvirtue of its increased activity. Therefore, nitrile hydratase of thebiocatalyst composition, and in particular formed during storage of thebiocatalyst, is new. Also, the biocatalyst does not produce the malodours associated with putrefaction during the storage period.

Preferably the storage method allows the biocatalyst to be stored for atleast two days and more preferably one or more weeks. In particular thebiocatalyst may be stored from three to twenty eight days, for example 3to 14 days.

The presence of fermentation broth components such as urea are notessential to the composition or the storage method of this aspect ofinvention. Where fermentation broth components are present, this may beurea or a urea derivative. The urea derivative can be for example analkyl derivative of urea.

Urea or the urea derivative could be present in the biocatalystcomposition through its inclusion in the fermentation mixture. In oneform of the invention the composition or storage medium containing thebiocatalyst may be deoxygenated and contain fermentation brothcomponents such as urea.

A particularly advantageous feature of this aspect of the invention isthat it is no longer necessary to separate the biocatalyst from thefermentation mixture in which it was cultured. This is of significantvalue since it avoids the requirement for an additional processing step.Therefore the composition may also comprise a fermentation mixture,which is then stored. In the method of storing the biocatalyst, we findthat this may also be achieved in the presence of a fermentation mixturewithout any detrimental effects on the activity of the enzyme. This thenallows the fermentation broth to be used immediately to catalyse thereaction, or to allow it to be stored for several days or even weekswithout detriment whilst the bioconversion step is being carried outalso over a period of several days, thus ensuring a constant supply ofreadily available biocatalyst without need for additional processingsteps thus simplifying and reducing the cost of the bioconversion step.

The biocatalyst may conveniently be stored at temperatures above itsfreezing point. Typically the biocatalyst may be stored at ambienttemperatures, for instance up to 30 or 40° C. However, the advantage ofthe present method is that the biocatalyst may be stored at ambienttemperatures without any special precautions for monitoring andcontrolling the temperature. Preferably the biocatalyst is stored at atemperature between 4 and 30 or 40° C., more preferably between 5 and25° C., such as between 10 and 25° C. and in particular 15 to 25° C.

According to a further aspect of the present invention we provide amethod of producing an amide by contacting the corresponding nitrile bya nitrile hydratase, in which the biocatalyst is part of a compositionor stored in the form of a non-actively growing free cell microorganismin a storage medium in which the composition or storage medium comprisesfermentation broth, and the biocatalyst is (or is obtainable from) themicroorganism Rhodococcus rhodochrous strain NCIMB 41164 or a mutantthereof.

Thus in accordance with this aspect of the invention the biocatalyst mayhave been held in an environment containing oxygen or held in anoxygen-free environment. It may or may not contain residual fermentationbroth components such as urea prior to commencing the conversion of thenitrile. This may be resulting from storing the biocatalyst inaccordance with the storage aspect of the present invention oralternatively provided as a composition in accordance with the presentinvention.

As given previously it is not necessary to remove the biocatalyst fromthe fermentation mixture in which the biocatalyst has been prepared.Thus in a preferred form the environment in which the biocatalyst isheld also contains components of a fermentation broth. Therefore abiocatalyst composition containing components of a fermentation brothcan be combined with a nitrile which is then hydrated to thecorresponding amide. We have found surprisingly that in contrast toprevious knowledge, for instance in U.S. Pat. No. 5,567,608 states thatimmobilisation of the biocatalyst is preferable to prevent elution ofimpurities from the biocatalyst into the reaction product, that theinclusion of fermentation broth in the reaction mixture does not affectthe quality of the final product and this aspect is described in ourco-filed UK application 0327901.5, identified by case numberBT/3-22349/P1.

The fermentation mixture will comprise essential components for allowingmicroorganisms to be grown and sustained. In general the mixture will atleast contain a carbon source, nitrogen source and various nutrients.This may include a saccharide for instance a monosaccharide such asglucose or other sugar or a disaccharide or polysaccharide, ammoniumsalts, complex medium components such as yeast extract and peptone,amino acids, vitamins, phosphate salts, potassium, sodium, magnesium andcalcium salts, trace elements such as iron, cobalt, manganese,copper,zinc and the like. These and other ingredients can be included in thefermentation mixture at concentrations suitable for the particularmicroorganism. It is known that fermentations can be subject to changesin the productivity of the biocatalyst and the fermentation broth may beused at different stages of growth and so it is important to be able tostore the biocatalyst after production in such a way.

We find that the activity of the biocatalyst does not diminishsignificantly during the reaction for a prolonged period. Consequentlythe biocatalyst may be replaced less frequently. Preferably thebiocatalyst is used for a period of at least 2 days and losessubstantially no activity over that period.

Generally the catalysis of the reaction using nitrile hydratase enablesthe nitrile to be converted into the corresponding amide in a singlestep. This process is of particular value when the nitrile isacrylonitrile and the amide is acrylamide. It is desirable to carry outthis conversion step several times using a singlebatch of biocatalystfrom which portions are removed over a period of several days to carryout several reactions where nitrile is converted to amide. Thus, it isimportant to be able to store the biocatalyst as inexpensively aspossible without detriment to the catalyst whilst the bioconversion stepis carried out simultaneously. So in effect one batch of biocatalyst canbe stored ready for use to make several batches of for instanceacrylamide. Several batches could be from 5 to 10 or more batches, even15 to 20 batches.

In a further aspect of the invention we have found a way of improvingthe biocatalytic activity of a microorganism. The microorganism would becultured in a culture medium that comprises urea or a derivative ofurea. However, urea or the derivative of urea is introduced into theculture medium at least six hours after the start of growth of themicroorganism. Normally the culture medium is substantially free of ureaor the urea derivative for at least the first six hours of culturing themicroorganism and thereafter urea or a urea derivative is added to theculture medium. As indicated previously by substantially free we meanthat the culture medium contain less than 0.2 g/l, usually less than 0.1g/l and may contain no urea or the urea derivative. Preferably theculture medium is substantially free of urea or the urea derivative forat least 12 hours and sometimes at least 24 hours. However, in order tomaximise the biocatalytic activity it is preferred to introduce the ureaor the urea derivative within 48 hours of culturing.

The biocatalytic activity can be established in terms of enzyme activityas described herein.

Preferably the microorganism is capable of producing a nitrilehydratase. Suitably a biocatalyst comprising such a microorganism can beused to prepare amides from the corresponding nitrile by a hydrationprocess in which nitrile hydratase catalyses the reaction. The culturingof the microorganism by delayed introduction of urea or urea derivativeprovides increased nitrile hydratase activity particularly suitable forthis reaction. The process is particularly suitable for the preparationof (meth) acrylamide from (meth) acrylonitrile. Such a process may becarried out as described herein. In addition the biocatalyst may berecycled and reused.

It is particularly desirable that the microorganism is of theRhodococcus genus, preferably a Rhodococcus rhodochrous species,especially Rhodococcus rhodochrous NCIMB 41164.

The following examples provide an illustration of how to carry out theinvention.

EXAMPLE 1

Rhodococcus rhodochrous NCIMB 41164 was isolated from soil using anenrichment culture technique and it was grown on a medium containing thefollowing constituents (g/l): KH₂PO₄, 7.0; KH₂PO₄, 3.0; peptone, 5.0;yeast extract, 3.0; glucose, 5.0; MgSO₄, 0.5; trace metals solution, 5ml; acetonitrile, 20 ml. The pH was adjusted to 7.2. The nitrilehydratase activity was 4,000 μmol min⁻¹/g dry cells at 15° C. after 3days growth at 28° C.

EXAMPLE 2

(1) Rhodococcus rhodochrous NCIMB 41164 was grown in a 2L baffledErlenmeyer flask containing 400 mL culture medium containing thefollowing constituents (g/L): dipotassium hydrogen phosphate 0.7;Potassium hydrogen phosphate 0.3; glucose 10.0; peptone, 1.0; yeastextract 3.0; magnesium sulphate heptahydrate 0.5; Urea 5.0; cobaltchloride hexahydrate 0.01; tap water to 1L. The pH of the medium wasadjusted to pH 7.2. The culture was grown at 28° C. for 5 days afterwhich the nitrile hydratase activity was 47,900 μmol min⁻¹/g at 15° C.

(2) (a) Rhodococcus rhodochrous NCIMB 41164 was grown in the mediumdescribed in (1) except that peptone was omitted.

(b) Rhodococcus rhodochrous NCIMB 41164 was grown in the mediumdescribed in (2a) except that peptone was ommitted as was urea. Theorganism was cultured for 24 hours and then 5 g/L urea was added to theculture which was grown for a further 5 days.

(C) Rhodococcus rhodochrous NCIMB 41164 was grown in the mediumdescribed in (2a) except that urea was not included in the medium. Theorganism was cultured for 48 hours and then 5 g/L urea was added to theculture which was grown for a further 4 days.

(d) Rhodococcus rhodochrous NCIMB 41164 was grown in the mediumdescribed in (2a) except that urea was not included in the medium. Theorganism was cultured for 6 days.

Samples were taken from the four cultures described above at time=1, 2,3 and 6 days after growth commenced. The nitrile hydratase activitieswere measured at 15° C., see table 1. TABLE 1 Urea addition NitrileHydratase Activity μmol min⁻¹/mg dry cells time (days) T = 1 day T = 2days T = 3 days T = 6 days 0 9.1 24.2 24.8 37.6 1 1.0 21.6 49.3 41.3 2ND ND 15.1 15.3 None added 0.94 ND 0.46 0.98ND not determined

EXAMPLE 3

(1) Rhodococcus rhodochrous NCIMB 41164 was grown in a 280L fermentercontaining 180 L culture medium containing the following constituents(g/L): diPotassium hydrogen phosphate 0.7; Potassium hydrogen phosphate0.3; glucose 2.0; yeast extract 3.0; magnesium sulphate heptahydrate0.5; cobalt chloride hexahydrate 0.01;. The pH of the medium wasadjusted to pH 7.2. The culture was grown at 30° C. for 3 days. Urea wasadded to the culture after 17 h. The nitrile hydratase activity wasmeasured (at 30° C.) periodically. 22 h after the urea was added theactivity was approximately 176,000 μmol min⁻ ¹/g at 30° C. and after afurther 9 h the activity had increased to 323, 000 μmol min⁻¹/g.

(2) 625 g of water was charged to the reactor to which Rhodococcusrhodochrous NCIMB 41164 was added. The mixture was heated to 25° C.Acrylonitrile 375 g was fed to the reactor at a rate to maintain theconcentration at 2% (w/w). After 175 minutes all of the acrylonitrilehad been converted to acrylamide to a final concentration ofapproximately 50% (w/w).

(3) The cells from 2 were recovered by centrifugation and they weresuspended in 625 g water. This suspension was stored at 4° C. for 3 daysprior to re-charging to the reactor. The procedure described in 5 wasfollowed and again after 175 minutes all of the acrylonitrile wasconverted to acrylamide.

(4) The cells from 3 were treated as described in 3 above except theywere stored for 2 days prior to re-use. Again 50% acrylamide wassynthesised. The acrylic acid concentrations measured for the batches ofacrylamide generated in example 32-4)(5-7 are shown in Table 2. TABLE 2Acrylic acid concentrations measured in each of the acrylamide batchesExample number Acrylic Acid Concentration (ppm) 3-2 5650 3-3 102 3-4None detected (<10 ppm)

EXAMPLE 4

(1) Rhodococcus rhodochrous NCIMB 41164 was grown in a 280L fermentercontaining 180 L culture medium containing the following constituents(g/L): dipotassium hydrogen phosphate 0.7; Potassium hydrogen phosphate0.3; glucose 1.0; yeast extract 3.0; magnesium sulphate heptahydrate0.5; cobalt chloride hexahydrate 0.01; urea, 5.0. The pH of the mediumwas adjusted to pH 7.2. The culture was grown at 30° C. for 3 days.

25L of the fermentation broth was degassed with nitrogen for 20 minutesprior to storage at ambient temperature, which was approx. 5° C. for 3½days. The nitrile hydratase activity was measured 15 h after harvestingand it was found to be 242,000 U/g at 25° C. When the NH activity wasre-measured 3 days later it was found to be 293,000 U/g.

EXAMPLE 5

Rhodococcus rhodochrous NCIMB 41164 was grown in a 2 L Erlenmeyer flaskfor 5 days at 28° C. with shaking at 180 rpm in a culture mediumcontaining the following constituents in g/L: dipotassium hydrogenphosphate 0.7; Potassium hydrogen phosphate 0.3; glucose 10.0; yeastextract 3.0; urea 5.0; magnesium sulphate heptahydrate 0.5; cobaltchloride hexahydrate 0.01;. The pH of the medium was adjusted to pH 7.2.The culture broth was divided into two portions, one half of which wasdeoxygenated using nitrogen. Portions of both the deoxygenated and theoxygenated culture broth were incubated at 4, 15 and 25° C. for 1 week.The nitrile hydratase activity of the portions was measuredperiodically.

The results of the nitrile hydratase assays are shown in Table 3. Theresults are given in U/mg dry cells TABLE 3 Incubation Time (days) temp.0 1 2 3 5 7 4° C. (O2) 140 286 232 267 257 4° C. 274 214 293 (degassed)15° C. (O2) 15° C. 140 218 (degassed) 25° C. (O2) 140 143 25° C. 154 230(degassed)

It can be seen from the results in Example 5 that the biocatalyst can bestored effectively at ambient temperatures. Furthermore it can be seenthat the nitrile hydratase activity increased on this occasion onstorage in comparison to day zero.

EXAMPLE 6

Defrosted cells of Rhodococcus rhodochrous NCIMB 41164 were resuspendedin water. The nitrile hydratase activity was measured over a period of 1week. The relative nitrile hydratase activities measured are shown inTable 4. TABLE 4 Relative nitrile hydratase activity (%) Time (days) 4°C. 15° C. 25° C. 0 100 100 100 1 66 64 66 2 78 77 76 5 72 72 74 7 68 7473

The results in Table 4 show that the activity did not decrease at any ofthe temperatures of storage between the 1 and 7 day incubation period.

EXAMPLE 7

(1) Rhodococcus rhodochrous NCIMB 41164 was grown in a 0.5L baffledErlenmeyer flask containing 100 mL culture medium containing thefollowing constituents (g/L): diPotassium hydrogen phosphate 0.7;Potassium hydrogen phosphate 0.3; glucose 10.0; yeast extract 3.0;magnesium sulphate heptahydrate 0.5; Urea 5.0; cobalt chloridehexahydrate 0.01; tap water to 1L. The pH of the medium was adjusted topH 7.2. The culture was grown at 30° C. for 4 days. The nitrilehydratase activity was measured at 25° C. after 2,3 and 4 days growth.

(2) (a) Rhodococcus rhodochrous NCIMB 41164 was grown in the mediumdescribed in (1) except that the urea was replaced by dimethylurea. (b)Rhodococcus rhodochrous NCIMB 41164 was grown in the medium described in(1) except that the urea was replaced by ethylurea.

(c) Rhodococcus rhodochrous NCIMB 41164 was grown in the mediumdescribed in (1) except that 2.5 g/l urea and 2.5 g/l dimethylurea wereadded to the medium in place of the 5 g/l urea.

(d) Rhodococcus rhodochrous NCIMB 41164 was grown in the mediumdescribed in (1) except that 2.5 g/l urea and 2.5 g/l ethylurea wereadded in place of the 5 g/l urea.

The nitrile hydratase activities are shown in Table 5 TABLE 5 Nitrilehydratase activity (μmol/min/g dry cells) Urea compound 2 days 3 days 4days urea 6,800 34,800 123,200 Dimethylurea 14,600 73,800 97,600Ethylurea 14,500 110,100 not determined. Urea + 7,400 27,000 19,400dimethylurea Urea + ethylurea 6,000 6,900 73,850

1. A microorganism which is Rhodococcus rhodochrous strain NCIMB 41164or a mutant thereof.
 2. A method of culturing the microorganismRhodococcus rhodochrous strain NCIMB 41164 or mutant thereof in aculture medium that contains urea or a derivative of urea.
 3. A methodaccording to claim 2 in which urea or urea derivative is introduced intothe culture medium at least six hours after the start of growth of themicroorganism.
 4. A method according to claim 2 in which the culturemedium contains less than 0.2 g/l urea or the urea derivative for atleast the first 6 hours of culturing the microorganism and thereafterurea or the urea derivative is added to the culture medium.
 5. A methodaccording to claim 2 in which the culture medium contains less than 0.2g/l urea or the urea derivative for at least the first 12 hours ofculturing the microorganism and thereafter urea or the urea derivativeis added to the culture medium.
 6. A method according to claim 2 inwhich urea or the urea derivative is added to the culture medium within48 hours of culturing.
 7. A nitrile hydratase obtainable from amicroorganism which is Rhodococcus rhodochrous strain NCIMB 41164 or amutant thereof.
 8. A process of preparing an amide from thecorresponding nitrile wherein the nitrile is subjected to a hydrationreaction in an aqueous medium in the presence of a biocatalyst selectedfrom the group consisting of a microorganism which is a Rhodococcusrhodochrous strain NCIMB 41164, a mutant thereof and a nitrile hydrataseobtained from Rhodococcus rhodochrous strain NCIMB 41164 or a mutantthereof.
 9. A process according to claim 8 in which the amide is(meth)acrylamide.
 10. A process according to claim 9 in which thebiocatalyst is introduced into an aqueous medium and (meth)acrylonitrileis fed into the aqueous medium such that the concentration of(meth)acrylonitrile in the aqueous medium is maintained at up to 6% byweight.
 11. A process according to claim 10 in which the reactioncontinues until the concentration of acrylamide is between 30 and 55% byweight.
 12. A process according to claim 8 in which the biocatalyst isrecycled and reused.
 13. A method of improving the biocatalytic activityof a microorganism, in which the microorganism is cultured in a culturemedium that comprises urea or a derivative of urea, wherein urea or thederivative of urea is introduced into the culture medium at least 6hours after the start of growth of the microorganism.
 14. A methodaccording to claim 13 in which the culture medium contains less than 0.2g/l urea or the derivative of urea for at least the first 6 hours ofculturing the microorganism and thereafter urea or the derivative ofurea is added to the culture medium.
 15. A method according to claim 13in which the culture medium contains less than 0.2 g/l urea or thederivative of urea for at least the first 12 hours of culturing themicroorganism and thereafter urea or the derivative of urea is added tothe culture medium.
 16. A method according to claim 13 in which urea orthe urea derivative is added to the culture medium within 48 hours ofculturing.
 17. A method according to claim 13 in which the microorganismis capable of producing a nitrile hydratase.
 18. A method according toclaim 13 in which the microorganism is of the Rhodococcus genus.
 19. Aprocess of preparing an amide from the corresponding nitrite wherein thenitrite is subjected to a hydration reaction in an aqueous medium in thepresence of a biocatalyst selected from the group consisting of amicroorganism which is capable of producing a nitrile hydratase, whereinthe microorganism has been cultured by the method according to claim 13.20. A process according to claim 19 in which the amide is (meth)acrylamide.
 21. A process according to claim 19 in which the biocatalystis introduced into an aqueous medium and (meth)acrylonitrile is fed intothe aqueous medium such that the concentration of (meth)acrylonitrile inthe aqueous medium is maintained at up to 6% by weight.
 22. A processaccording to claim 21 in which the reaction continues until theconcentration of acrylamide is between 30 and 55% by weight.
 23. Aprocess according to claim 19 in which the biocatalyst is recycled andreused.
 24. An aqueous composition comprising a biocatalyst that is oris obtained from the microorganism Rhodococcus rhodochrous strain NCIMB41164 or a mutant thereof and wherein the biocatalyst is in the form ofa non-actively growing free cell microorganism.
 25. A method of storingthe biocatalyst that is or is obtained from the microorganismRhodococcus rhodochrous strain NCIMB 41164 or a mutant thereof in theform of a non-actively growing free cell microorganism, in an aqueousstorage medium.
 26. A method according to claim 25 in which thebiocatalyst is stored at a temperature above its freezing point.
 27. Amethod according to claim 25 in which the biocatalyst is stored for aperiod of at least two days.
 28. A composition obtained by the methodaccording to claim
 25. 29. A nitrile hydratase obtained from thecomposition according to claim 24 or obtained by the method of storingthe biocatalyst that is or is obtained from the microorganismRhodococcus rhodochrous strain NCIMB 41164 or a mutant thereof in theform of a non-actively growing free cell microorganism, in an aqueousstorage medium.
 30. A method of producing an amide by contacting thecorresponding nitrile with a nitrile hydratase, wherein the nitrilehydratase is obtained from a composition according to claim 24 orobtained by a method storing the biocatalyst that is or is obtained fromthe microorganism Rhodococcus rhodochrous strain NCIMB 41164 or a mutantthereof in the form of a non-actively growing free cell microorganism,in an aqueous storage medium.
 31. A method according to claim 30 inwhich the amide is (meth)acrylamide.